Nicholas Chiorazzi

Chronic lymphocytic leukemia cells recognize conserved epitopes associated with apoptosis and oxidation.

Chronic lymphocytic leukemia (CLL) represents the outgrowth of a CD5+ B cell. Its etiology is unknown. The structure of membrane Ig on CLL cells of unrelated patients can be remarkably similar. Therefore, antigen binding and stimulation could contribute to clonal selection and expansion as well as disease promotion. Initial studies suggest that CLL mAbs bind autoantigens. Since apoptosis can make autoantigens accessible for recognition by antibodies, and also create neo-epitopes by chemical modifications occurring naturally during this process, we sought to determine if CLL mAbs recognize autoantigens associated with apoptosis. In general, ~60% of CLL mAbs bound the surfaces of apoptotic cells, were polyreactive, and expressed unmutated IGHV. mAbs recognized two types of antigens: native molecules located within healthy cells, which relocated to the external cell surface during apoptosis; and/or neoantigens, generated by oxidation during the apoptotic process. Some of the latter epitopes are similar to those on bacteria and other microbes. Although most of the reactive mAbs were not mutated, the use of unmutated IGHV did not bestow autoreactivity automatically, since several such mAbs were not reactive. Particular IGHV and IGHV/D/J rearrangements contributed to autoantigen binding, although the presence and degree of reactivity varied based on specific structural elements. Thus, clonal expansion in CLL may be stimulated by autoantigens occurring naturally during apoptosis. These data suggest that CLL may derive from normal B cells whose function is to remove cellular debris, and also to provide a first line of defense against pathogens.

Intraclonal complexity in chronic lymphocytic leukemia: fractions enriched in recently born/divided and older/quiescent cells.

The failure of chemotherapeutic regimens to eradicate cancers often results from the outgrowth of minor subclones with more dangerous genomic abnormalities or with self-renewing capacity. To explore such intratumor complexities in B-cell chronic lymphocytic leukemia (CLL), we measured B-cell kinetics in vivo by quantifying deuterium (2H)-labeled cells as an indicator of a cell that had divided. Separating CLL clones on the basis of reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and cluster designation 5 (CD5) revealed that the CXCR4dimCD5bright (proliferative) fraction contained more 2H-labeled DNA and hence divided cells than the CXCR4brightCD5dim (resting) fraction. This enrichment was confirmed by the relative expression of two cell cycle-associated molecules in the same fractions, Ki-67 and minichromosome maintenance protein 6 (MCM6). Comparisons of global gene expression between the CXCR4dimCD5bright and CXCR4brightCD5dim fractions indicated higher levels of pro-proliferation and antiapoptotic genes and genes involved in oxidative injury in the proliferative fraction. An extended immunophenotype was also defined, providing a wider range of surface molecules characteristic of each fraction. These intraclonal analyses suggest a model of CLL cell biology in which the leukemic clone contains a spectrum of cells from the proliferative fraction, enriched in recently divided robust cells that are lymphoid tissue emigrants, to the resting fraction enriched in older, less vital cells that need to immigrate to lymphoid tissue or die. The model also suggests several targets preferentially expressed in the two populations amenable for therapeutic attack. Finally, the study lays the groundwork for future analyses that might provide a more robust understanding of the development and clonal evolution of this currently incurable disease.

Implications of new prognostic markers in chronic lymphocytic leukemia

Several prognostic markers based on genetic, phenotypic, and molecular characteristics of chronic lymphocytic leukemia (CLL) B cells have emerged in the past decade. The clinical utility of these newer prognostic indicators, alone or in combination with each other and other clinical predictive systems, is still being determined. This chapter attempts to define biologic and molecular underpinnings of 3 sets of prognostic indicators in CLL: genetic abnormalities quantified by FISH and/or defined by exploratory sensitive molecular techniques, expression of specific proteins in or on CLL cells (ie, CD38, CD49d, and ZAP-70), and the IGHV mutation status of a CLL clone. Although not demonstrated conclusively, each probably reflects the biologic properties of the leukemic cells of individual CLL patients. This reflection may be direct, indicating a specific property of the CLL cell itself, or indirect, representing how the CLL cell interacts with the hosts microenvironment. The new tyrosine kinase inhibitors that are currently in clinical trials support this interpretation. These and other biology-based indicators of patient clinical course and outcome can be used as starting points from which to understand and treat CLL.

VH gene usage by family members affected with chronic lymphocytic leukaemia

The excess risk of chronic lymphocytic leukaemia (CLL) in the first‐degree relatives of affected patients suggests that familial CLL might constitute a useful model to study the pathogenesis of this disease, as has been demonstrated in numerous other neoplastic disorders. Previous studies have shown non‐random utilization of immunoglobulin genes in CLL, some germline in sequence and others containing numerous somatic mutations. To investigate whether familial cases of CLL exhibit similarities in the composition of the B‐cell receptor repertoire to the pattern expressed by CLL patients as a whole, we have studied 25 CLL patients belonging to 12 different families (four French and eight Italian), each of which contained at least two affected members.

BTK inhibition results in impaired CXCR4 chemokine receptor surface expression, signaling and function in chronic lymphocytic leukemia.

Bruton’s tyrosine kinase (BTK) is involved in the regulation of B-cell growth, migration and adhesion. The importance of BTK in cell trafficking is emphasized by the clonal contraction proceeded by lymphocytosis typical for the enzyme inhibitor, ibrutinib, in B-cell malignancies, including chronic lymphocytic leukemia (CLL). Here, we investigated BTK regulation of leukemic B-cell trafficking in a mouse model of aggressive TCL1 CLL-like disease. Inhibiting BTK by ibrutinib reduced surface membrane (sm) levels of CXCR4 but not CXCR5, CD49d and other adhesion/homing receptors. Decreased smCXCR4 levels resulted from blocking receptor signal transduction, which in turn aborted cycling from and to the membrane. This resulted in rapid re-distribution of CLL cells from spleens and lymph nodes into the circulation. CLL cells with impaired smCXCR4 from BTK inhibition failed to home to spleens. These functional changes mainly resulted from inhibition of CXCR4 phosphorylation at Ser339, mediated directly by blocking BTK enzymatic activity and indirectly by affecting the function of downstream targets PLCγ2 and PKCμ, and eventually synthesis of PIM-1 and BTK itself. Our data identify CXCR4 as a key regulator in BTK-mediated CLL-cell retention and have elucidated a complex set of not previously described mechanisms responsible for these effects.

The Human Marginal Zone B Cell

Abstract: This study describes the features of the marginal zone (MZ) B cells of human tonsils and spleens and compares them with those of the follicular mantle (FM) B cells from the same tissues. The two B cell subpopulations displayed marked differences in phenotype, in response capacity to T cell‐independent antigens and polyclonal B cell activators, and in presentation of antigens to T cells. FM B cells expressed surface CD5, and hence should be considered as B1 cells by current nomenclature. Fractionation of MZ B cells according to the presence or absence of surface IgD revealed the presence of two subsets. These subsets were characterized by different properties, including the presence of Ig VH gene mutations and the response capacity to TI‐2 antigens, this latter property being associated with IgD‐positive cells. Comparison of the data with those reported for mice revealed that human MZ B cells had strong analogies with both the murine MZ and B1 cells. In contrast, human B1 cells (that is, CD5‐positive FM cells) were considerably different, an observation that should prompt further studies. Indeed, B cells with characteristics analogous to those of murine B1 cells were detected in small but definite proportions in the peripheral blood and tonsils. If the current distinction into B1 and B2 cells has to be maintained also for humans, it is likely that only these CD5‐positive cells rather than the FM B cells should be called B1 cells.

Simultaneous flow cytometric analysis of cell surface markers and telomere length: analysis of human tonsilar B cells

Telomere Flow FISH is a recently developed method which allows the measurement of telomere length in purified subsets of cells using flow cytometry. However, the harsh conditions required for flow FISH have precluded its use with conventional cell surface staining, thus limiting its utility for large scale clinical studies. We have now developed a method which permits simultaneous analysis of cell surface markers along with telomere length estimation by flow cytometry. This new assay employs the covalent crosslinking of monoclonal antibodies conjugated with a heat stable fluorochrome to the cell surface prior to flow FISH. Using this technique we have confirmed that human germinal center B cells (IgD(-)/CD38(+)) have dramatically longer telomeres than pre-germinal center founder B cells (IgD(+)/CD38(+)). This approach simplifies the analysis of complex cell populations and will facilitate widespread investigation of telomere length in health and disease states.

Alternative splicing of CD79a (Ig-αmb-1) and CD79b (Ig-βB29) RNA transcripts in human B cells

The CD79a (Ig-alpha/mb-1) and CD79b (Ig-beta/B29) molecules form a membrane heterodimer that is non-covalently associated with surface membrane immunoglobulin and is the major signaling component of the B cell antigen receptor complex. We have defined variant RNA transcripts for both CD79a (Ig-alpha/mb-1) and CD79b (Ig-beta/B29) which appear to arise by alternative splicing. These splice variants are predicted to encode truncated forms of these molecules that result in the deletion of the entire extracellular Ig-like domain of CD79b and of a major portion of the extracellular domain of CD79a. The presence of these short transcripts in a variety of human B cells and B cell lines was established by an RNAse protection assay. The definition of these variant transcripts provides a basis for a continuing effort to define variant protein products of CD79a and CD79b and examine their role in B cell physiology.

Maintenance of B lymphocyte-related clones in the cerebrospinal fluid of multiple sclerosis patients.

A longitudinal study of Ig V gene segments utilized by B cells from the cerebrospinal fluid (CSF) of two patients with multiple sclerosis (MS) was carried out using RT‐PCR methodologies. One patient with a relapsing‐remitting (RR)‐MS was investigated at onset and at relapse, 1 year later. A patient with secondary‐progressive (SP)‐MS was tested 9 and 13 years after disease onset. Sequenceanalyses of VHDJH segments bearing VH3 and VH4 that were obtained from Cγ cDNA genes demonstrated a substantial proportion of shared clones in the samples taken at different times; these clones were identical or closely related, i.e. had the same third complementary determining region (CDR) of the H chain variable region gene (HCDR3) with different mutations in the VH segment. Collectively, these data demonstrate that in MS patients there is a strong selective pressure, which could be exerted by antigen (or autoantigen) stimulation, for the maintenance and partial diversification of certain VHDJH Cγ sequences.

Th17 and non-Th17 interleukin-17-expressing cells in chronic lymphocytic leukemia: delineation, distribution, and clinical relevance

Background The levels and clinical relevance of Th17 cells and other interleukin-17-producing cells have not been analyzed in chronic lymphocytic leukemia. The objective of this study was to quantify blood and tissue levels of Th17 and other interleukin-17-producing cells in patients with this disease and correlate blood levels with clinical outcome. Design and Methods Intracellular interleukin-17A was assessed in blood and splenic mononuclear cells from patients with chronic lymphocytic leukemia and healthy subjects using flow cytometry. Interleukin-17A-producing cells were analyzed in formalin-fixed, paraffin-embedded spleen and lymph node sections using immunohistochemistry and immunofluorescence. Results The absolute numbers of Th17 cells in peripheral blood mononuclear cells and the percentages of Th17 cells in spleen cell suspensions were higher in patients with chronic lymphocytic leukemia than in healthy subjects; in six out of eight paired chronic lymphocytic leukemia blood and spleen sample comparisons, Th17 cells were enriched in spleen suspensions. Circulating Th17 levels correlated with better prognostic markers and longer overall survival of the patients. Two “non-Th17” interleukin-17-expressing cells were identified in chronic lymphocytic leukemia spleens: proliferating cells of the granulocytic lineage and mature mast cells. Granulocytes and mast cells in normal spleens did not express interleukin-17. Conversely, both chronic lymphocytic leukemia and healthy lymph nodes contained similar numbers of interleukin-17+ mast cells as well as Th17 cells. Conclusions Th17 cells are elevated in chronic lymphocytic leukemia patients with better prognostic markers and correlate with longer survival. Furthermore, non-Th17 interleukin-17A-expressing cells exist in chronic lymphocytic leukemia spleens as maturing granulocytes and mature mast cells, suggesting that the microenvironmental milieu in leukemic spleens promotes the recruitment and/or expansion of Th17 and other IL-17-expressing cells. The pathophysiology of Th17 and non-Th17-interleukin-producing cells in chronic lymphocytic leukemia and their distributions and roles in this disease merit further study.